We used a high-performance fluorescence imaging system to visualize rapid changes in intracellular free Ca2+ concentration ([Ca2+]i) evoked by focal applications of extracellullar ATP to the hair bundle of outer hair cells (OHCs) - the sensory-motor receptors of the cochlea. Simultaneous recordings of the whole-cell current and Calcium Green-1 fluorescence showed a two-component increase in [Ca2+]i. After an initial entry of Ca2+ through the apical membrane, a second and larger, InsP3-gated, [Ca2+]i surge occurred at the base of the hair bundle. Electron microscopy of this intracellular Ca2+ release site showed that it coincides with the localization of a unique system of endoplasmic reticulum (ER) membranes and mitochondria known as Hensens body. Using confocal immunofluorescence microscopy, we showed that InsP3 receptors share this location. Consistent with a Ca2+-mobilizing second messenger system linked to ATP- P2 receptors we also determined that an isoform of G proteins is present in the stereocilia. Voltage-driven cell shape changes and nonlinear capacitance were monitored before and after ATP application, showing that the ATP-evoked [Ca2+]i rise did not interfere with the OHC electromotility mechanism. This second messenger signaling mechanism bypasses the Ca2+- clearance power of the stereocilia and transiently elevates [Ca2+]i at the base of the hair bundle, where it can potentially modulate the action of unconventional myosin isozymes involved in maintaining the hair bundle integrity and potentially influence mechano-transduction. - hair sensory cells, mechano transduction, electromotility, cochlear amplifier, organ of Corti, outer hair cells, purinergic receptors.